High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli.
Protein Expr Purif
; 90(1): 20-6, 2013 Jul.
Article
em En
| MEDLINE
| ID: mdl-23628981
ABSTRACT
Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Escherichia coli
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Fator Inibidor de Leucemia
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article